사료의 발효가 영양 성분 및 반추위 in vitro 발효에 미치는 영향

이슬(Seul Lee),백열창(Youlchang Baek),방한태(Han-Tae Bang) 한국산학기술학회 2022 한국산학기술학회논문지 Vol.23 No.11

발효사료는 인공적인 제조 과정을 통해 미생물 발효한 사료를 의미하며 영양소, 소화율, 기호성, 안전성 측면에서 우수하다. 발효사료의 효능은 제조 조건 및 급이 환경에 따라 변이가 있어, 본 연구에서는 사료의 발효에 따라 영양 성분의 변화와 반추위 이용성을 평가하여 발효사료의 우수성을 검증하는 것을 목적으로 하였다. 본 시험은 발효에 따른 영양성분 변화와 반추위 발효 성상 변화를 분석하였고, 2 (발효 유무) × 2 (조사료 첨가 유무)의 요인설계를 실시하였다. 동결 건조된 박테리아 혼합물(Bacillus subtilis, Enterococcus faecium, Saccharomyces cerevisiae )과 한우용 배합사료를 이용하여 발효사료를 제조하였고, 반추위 in vitro 배양 시험을 통해 39 °C에서 24시간 배양하였다. 화학 성분 분석 결과, 발효사료의 crude protein (CP)은 2.6%p 증가하고, non-fiber carbohydrate (NFC)는 5.1%p 감소하였다. In vitro 배양 결과 조사료 첨가 여부에 관계없이 발효사료 처리구의 건물 소화율이 유의적으로 증가하였고(p <0.01), 총 volatile fatty acids (VFA)와 암모니아태 질소 함량은 유의적으로 감소하였으며(p < 0.01) 소화된 건물 당 메탄 발생량이 감소하는 경향을 보였다(p = 0.07). 따라서 발효사료를 한우에 급여할 시 CP가 증진되어 영양적인 측면에서 우수하고, 반추위 분해율이 증가하여 가축의 영양소 이용성을 높일 것으로 판단된다. Fermented feed refers to feed fermented by microorganisms through an artificial manufacturing process and is excellent in nutrients, digestibility, palatability, and safety. Since the efficacy of the feed varies and depends on manufacturing conditions and the feeding environment, this study was undertaken to verify the superiority of fermented feed by evaluating the changes in nutrient composition and ruminal degradability. Changes in the nutrient composition and in vitro ruminal degradability were evaluated using a 2 (non-fermented; fermented) × 2 (concentrate 100%; concentrate 50%, forage 50%) factor design. Fermented feed was prepared using a mixture of freeze-dried bacteria (Bacillus subtilis, Enterococcus faecium, Saccharomyces cerevisiae) and mixed feed for Korean cattle, and subjected to in vitro ruminal incubation culture for 24 hours at 39°C. The chemical composition analysis revealed that the crude protein (CP) of fermented feed increased by 2.6%p and non-fiber carbohydrate (NFC) decreased by 5.1%p. In vitro incubation resulted in the fermented feed showing relatively high dry matter digestibility (p < 0.01), and low total volatile fatty acids (VFA) and ammonia nitrogen content (p < 0.01). Moreover, the amount of methane generated per digested dry matter (p = 0.07) showed a decreasing tendency. In conclusion, we believe that fermented feed is advantageous in nutrition and rumen digestibility, which could increase the nutrient availability for Hanwoo cattle.

Methanogenic Archaeal Census of Ruminal Microbiomes

Seul Lee(이슬),Youlchang Baek(백열창),Jinwook Lee(이진욱),Minseok Kim(김민석) 한국산학기술학회 2020 한국산학기술학회논문지 Vol.21 No.7

본 연구의 목적은 Ribosomal Database Project에서 공적으로 활용 가능한 16S rRNA 유전자 시퀀스들의 메타분석을 통해 반추위 고세균의 계통발생 다양성을 조사하는 것이다. 총 8,416개의 시퀀스가 Ribosomal Database Project(출시버전 11, 업데이트 5)로부터 회수되었고, taxonomy tree를 구축하는데 사용되었다. Species 수준의 OTUs가 97% sequence 유사성 기준으로 QIIME 프로그램을 사용하여 분석되었다. 총 8,416개의 시퀀스 중에서 8,412개의 시퀀스는 총 3개의 문으로 분류되었고, 나머지 4개의 시퀀스는 어떤 알려진 문으로 분류되지 못했다. Euryarchaeota 는 가장 우점하는 문으로, 전체 고세균 시퀀스의 99.8%를 차지하였다. 이 중에서 차례로 Methanobrevibacter 가 65.4%, Methanosphaera 가 10.4%, Methanomassillicoccus 가 10.4%, Methanomicrobium 가 7.9%, Methanobacterium 가 1.9%, Methanimicrococcus 가 0.5%, Methanosarcina 가 0.1%, Methanoculleus 가 0.1%를 차지하였다. V2와 V3 영역으로 자른 7,544개의 시퀀스는 493개의 OTUs로 분류되었다. 총 493 OTUs 중에서 단지 17개만 우점하였고, 총 7,544 시퀀스 중 1% 이상을 차지하였다. 본 연구는 반추동물로 부터 메탄발생에 크게 기여하는 반추위 우점 메탄생성균 분석에 대한 향후 연구를 인도하는데 도움을 주고, 사료나 가축품종이 달라져도 이러한 메탄생성균을 억제하는 메탄저감제를 개발하는데 도움을 줄 것이다. The objective of the study was to undertake a phylogenetic diversity census of ruminal archaea based on a meta-analysis of 16S rRNA gene sequences that were publicly available in the Ribosomal Database Project. A total of 8,416 sequences were retrieved from the Ribosomal Database Project (release 11, update 5) and included in the construction of a taxonomy tree. Species-level operational taxonomic units (OTUs) were analyzed at a 97% sequence similarity by using the QIIME program. Of the 8,416 sequences, 8,412 were classified into one of three phyla; however, the remaining four sequences could not be classified into a known phylum. The Euryarchaeota phylum was predominant and accounted for 99.8% of the archaeal sequences examined. Among the Euryarchaeota, 65.4% were assigned to Methanobrevibacter, followed by Methanosphaera (10.4%), Methanomassillicoccus (10.4%), Methanomicrobium (7.9%), Methanobacterium (1.9%), Methanimicrococcus (0.5%), Methanosarcina (0.1%), and Methanoculleus (0.1%). The 7,544 sequences that had been trimmed to the V2 and V3 regions clustered into 493 OTUs. Only 17 of those 493 OTUs were dominant groups and accounted for more than 1% of the 7,544 sequences. These results can help guide future research into the dominant ruminal methanogens that significantly contribute to methane emissions from ruminants, research that may lead to the development of anti-methanogenic compounds that inhibit these methanogens regardless of diet or animal species.

Next Generation Sequencing을 통한 미생물 군집 분석의 축산분야 활용

김민석,백열창,오영균,Kim, Minseok,Baek, Youlchang,Oh, Young Kyoon 한국축산환경학회 2015 한국축산시설환경학회지 Vol.21 No.3

The objective of this study was to review application of next-generation sequencing (NGS) to investigate microbiome in the livestock sector. Since the 16S rRNA gene is used as a phylogenetic marker, unculturable members of microbiome in nature or managed environments have been investigated using the NGS technique based on 16S rRNA genes. However, few NGS studies have been conducted to investigate microbiome in the livestock sector. The 16S rRNA gene sequences obtained from NGS are classified to microbial taxa against the 16S rRNA gene reference database such as RDP, Greengenes and Silva databases. The sequences also are clustered into species-level OTUs at 97% sequence similarity. Microbiome similarity among treatment groups is visualized using principal coordinates analysis, while microbiome shared among treatment groups is visualized using a venn diagram. The use of the NGS technique will contribute to elucidating roles of microbiome in the livestock sector.

Effects of Sampling Techniques and Sites on Rumen Microbiome and Fermentation Parameters in Hanwoo Steers

( Jaeyong Song ),( Hyuck Choi ),( Jin Young Jeong ),( Seul Lee ),( Hyun Jung Lee ),( Youlchang Baek ),( Sang Yun Ji ),( Minseok Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.10

We evaluated the influence of sampling technique (cannulation vs. stomach tube) and site (dorsal sac vs. ventral sac) on the rumen microbiome and fermentation parameters in Hanwoo steers. Rumen samples were collected from three cannulated Hanwoo steers via both a stomach tube and cannulation, and 16S rRNA gene amplicons were sequenced on the MiSeq platform to investigate the rumen microbiome composition among samples obtained via 1) the stomach tube, 2) dorsal sac via rumen cannulation, and 3) ventral sac via rumen cannulation. A total of 722,001 high-quality 16S rRNA gene sequences were obtained from the three groups and subjected to phylogenetic analysis. There was no significant difference in the composition of the major taxa or alpha diversity among the three groups (p> 0.05). Bacteroidetes and Firmicutes represented the first and second most dominant phyla, respectively, and their abundances did not differ among the three groups (p> 0.05). Beta diversity principal coordinate analysis also did not separate the rumen microbiome based on the three sample groups. Moreover, there was no effect of sampling site or method on fermentation parameters, including pH and volatile fatty acids (p > 0.05). Overall, this study demonstrates that the rumen microbiome and fermentation parameters are not affected by different sampling techniques and sampling sites. Therefore, a stomach tube can be a feasible alternative method to collect representative rumen samples rather than the standard and more invasive method of rumen cannulation in Hanwoo steers.

Effect of commercially purified deoxynivalenol and zearalenone mycotoxins on microbial diversity of pig cecum contents

Reddy, Kondreddy Eswar,Kim, Minji,Kim, Ki Hyun,Ji, Sang Yun,Baek, Youlchang,Chun, Ju Lan,Jung, Hyun Jung,Choe, Changyong,Lee, Hyun Jeong,Kim, Minseok,Lee, Sung Dae Asian Australasian Association of Animal Productio 2021 Animal Bioscience Vol.34 No.2

Objective: Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins that frequently contaminate maize and grain cereals, imposing risks to the health of both humans and animals and leading to economic losses. The gut microbiome has been shown to help combat the effects of such toxins, with certain microorganisms reported to contribute significantly to the detoxification process. Methods: We examined the cecum contents of three different dietary groups of pigs (control, as well as diets contaminated with 8 mg DON/kg feed or 0.8 mg ZEN/kg feed). Bacterial 16S rRNA gene amplicons were acquired from the cecum contents and evaluated by next-generation sequencing. Results: A total of 2,539,288 sequences were generated with ~500 nucleotide read lengths. Firmicutes, Bacteroidetes, and Proteobacteria were the dominant phyla, occupying more than 96% of all three groups. Lactobacillus, Bacteroides, Megasphaera, and Campylobacter showed potential as biomarkers for each group. Particularly, Lactobacillus and Bacteroides were more abundant in the DON and ZEN groups than in the control. Additionally, 52,414 operational taxonomic units were detected in the three groups; those of Bacteroides, Lactobacillus, Campylobacter, and Prevotella were most dominant and significantly varied between groups. Hence, contamination of feed by DON and ZEN affected the cecum microbiota, while Lactobacillus and Bacteroides were highly abundant and positively influenced the host physiology. Conclusion: Lactobacillus and Bacteroides play key roles in the process of detoxification and improving the immune response. We, therefore, believe that these results may be useful for determining whether disturbances in the intestinal microflora, such as the toxic effects of DON and ZEN, can be treated by modulating the intestinal bacterial flora.

Analysis of Rumen Fermentation and Methanogen Levels in Response to Various Alfalfa Hay, Oat Hay, and Feed Concentrate Ratios

Seul Lee,Banji Kim,Chaehwa Ryu,Jinyoung Jeong,Byeonghyeon Kim,Youlchang Baek 한국초지조사료학회 2020 한국초지조사료학회지 Vol.40 No.4

This study aimed to analyze ruminal fermentation, methane emissions, and methanogen levels for different forage feed type and concentrate feed ratios. Alfalfa hay, oat hay, and a feed concentrate were used for in vitro fermentation experiments, at ratios of 9:1, 5:5, and 1:9 (forage:concentrate). After 24 h of incubation, rumen fermentation and methanogen level changes were evaluated. In the low forage treatments, the total gas, CH4, NH3-N, true dry matter digestibility, and total volatile fatty acid were higher than the other treatments, which were used as the parameters on which to assess rumen fermentation (P < 0.05). The feed ratio influenced the copy number for the total archaea and the genus Methanobrevibacter (P = 0.015, P = 0.010). The copy number result trend was like that for CH4 per digested dry matter (DDM). The PCR results and methanogen copy number analysis indicated that the composition of the methanogens affected the CH4 levels, not their copy number. The results of this study can be applied to predict rumen fermentation and methane emission patterns for cattle fed a variety of feedstuffs.

조사료와 농후사료의 급여 순서가 In vitro 반추위 발효성상, 총 가스 발생량과 메탄 발생량에 미치는 영향

이유경,이성실,성필남,이슬,백열창,김기현,이성대,천주란,지상윤,김정은,Lee, Yookyung,Lee, Sungsill,Seong, Pilnam,Lee, Seul,Baek, Youlchang,Kim, Kihyun,Lee, Sungdae,Chun, Julan,Ji, Sangyun,Kim, Jungeun 한국초지조사료학회 2021 한국초지조사료학회지 Vol.41 No.2

본 연구는 국내 고유종인 한우에서 1) 농후사료 급여 후 조사료 급여, 2) 조사료 급여 후 농후사료 급여, 3) TMR 급여의 총 세 가지 형태의 사료급여가 반추위 발효성상, 총 가스 및 메탄 발생량에 미치는 영향을 in vitro로 평가하기 위해 수행되었다. 사료 급여 형태에 따라 반추위 환경이 변화할 것으로 가설을 세우고, 옥수수 (M)와 티모시 (T)를 기질로 하여 반추위액의 24시간 in vitro 배양을 실시하였다. 따라서 시험 처리구는 총 6개로 구성되었다: M1, M2, M3, T1, T2, T3. 반추위 pH는 사료 급여방식 2에서 다른 처리구보다 높았고 (P < 0.001), 티모시 기질에서 옥수수 기질보다 높았다 (P < 0.001). 휘발성 지방산 (VFA; volatile fatty acid) 조사항목들 (Total VFA, acetate, propionate, butyrate, valerate)은 사료 급여방식 1에서 가장 높았고 급여방식 2에서 가장 낮았으며 (P < 0.001), 옥수수 기질에서 더 많이 생산되었다 (P < 0.001). 반추위액 내 acetate:propionate 비율은 사료 급여방식에 따른 효과는 없었으나 (P = 0.116), 티모시 기질이 옥수수 기질보다 더 높은 값을 나타냈다 (P < 0.001). 암모니아태 질소 (NH₃-N)는 사료 급여방식 1에서 다른 처리구보다 높았으며(P < 0.001), 티모시 기질에서 높은 수치를 보였다 (P < 0.001). 총 가스 및 메탄가스 발생량은 사료 급여방식 1에서 가장 높았고(P < 0.001), 옥수수 기질에서 더 많이 생산되었다 (P < 0.001). 메탄은 이산화탄소의 20배 이상의 지구온난화효과를 갖는 것으로 알려져 있고, 반추가축이 섭취한 에너지의 손실로 작용하고 있어 장내발효 메탄 발생량을 저감하는 것은 매우 중요한 과제라 할 수 있다. 반추가축에서 사료급여 방식에 따른 메탄 발생량 차이에 대한 결론을 얻기 위해서는 보다 다각적인 형태의 연구가 필요할 것으로 사료된다. The objective of this study was to evaluate effects of feeding methods on in vitro ruminal fermentation, total gas and methane production in Hanwoo steers. Six Hanwoo steers fitted with rumen cannula (430 ± 21 kg of body weight) were randomly assigned to one of three feeding systems: 1) feeding forage 1 hour after concentrate, 2) feeding concentrate 1 hour after forage, 3) feeding mixed ration. Rumen fluid sampled from each animals was incubated 24 hours with maize or timothy substrates in in vitro. Ruminal pH was increased in feeding method 2 or maize substrate than that of other methods or timothy substrate (P < 0.001). The production of total volatile fatty acid, acetate, propionate, butyrate, and valerate were increased when steers fed diets using feeding method 1 or rumen fluid was incubated with maize substrate (P < 0.001). Increased production of total gas and methane was observed in feeding method 1 and maize substrate compared to those of other methods or timothy substrate (P < 0.001). Due to the inconsistent results between ruminal fermentation and gas production in this study, further research is required to estimate effects of feeding method on enteric fermentation and gas production in in vivo.

Effects of amino acid composition in pig diet on odorous compounds and microbial characteristics of swine excreta

( Neeraja Recharla ),( Kihyun Kim ),( Juncheol Park ),( Jinyoung Jeong ),( Yongdae Jeong ),( Hyunjeong Lee ),( Okhwa Hwang ),( Jaehyoung Ryu ),( Youlchang Baek ),( Youngkyun Oh ),( Sungkwon Park ) 한국축산학회(구 한국동물자원과학회) 2017 한국축산학회지 Vol.59 No.12

Background: Major amino acids in pig diets are Lys, Met, Thr, and Trp, but little is known about the requirements for the other essential amino acids, especially on odorous compounds and microbial characteristics in feces of growing-finishing pigs. To this end, different levels of amino acid composition added to diets to investigate the effects of amino acid composition on microbial characteristics and odorous compounds concentration. Methods: A total eight (n = 8) barrows (Landrace × Yorkshire × Duroc) with an average bodyweight of 89.38 ± 3. 3 kg were individually fed diets formulated by Korean Feeding Standards 2007 (old version) or 2012 (updated with ideal protein concept) in metabolism crates with two replication. After 15-day adaptation period, fresh faecal samples were collected directly from pigs every week for 4 weeks and analysed for total volatile fatty acids (VFA), phenols and indoles by using gas chromatography. The nitrogen was determined by Kjeldahl method. Bacterial communities were detected by using a 454 FLX titanium pyrosequencing system. Results: Level of VFA tended to be greater in 2012 than 2007 group. Among VFAs, 2012 group had greater (p < 0.05) level of short chain fatty acids (SCFA) than control.Concentration of odorous compounds in feces was also affected by amino acid composition in pig diet. Levels of ammonium and indoles tended to be higher in 2012 group when compared with 2007 group.Concentration of phenols, p-cresol, biochemical oxygen demand, and total Kjeldahl nitrogen, however, were lower (P < 0.05) in 2012 treatment group compare to 2007. The proportion of Firmicute phylum were decreased, while the Bacteriodetes phylum proportion increased and bacterial genera includingCoprococcus, Bacillus, and Bacteroides increased (p < 0.05) in 2012 compare to 2007 group. Conclusion: Results from our current study indicates that well balanced amino acid composition reduces odor by modulating the gut microbial community. Administration of pig diet formulated with the ideal protein concept may help improve gut fermentation as well as reduce the odor causing compounds in pig manure.